Capturing the Kidney Transcriptome by Urinary Extracellular Vesicles-From Pre-Analytical Obstacles to Biomarker Research

Urinary extracellular vesicles (uEV) hold non-invasive RNA biomarkers for genitourinary tract diseases. However, missing knowledge about reference genes and effects of preanalytical choices hinder biomarker studies. We aimed to assess how preanalytical variables (urine storage temperature, isolation workflow) affect diabetic kidney disease (DKD)-linked miRNAs or kidney-linked miRNAs and mRNAs (kidney-RNAs) in uEV isolates and to discover stable reference mRNAs across diverse uEV datasets. We studied nine raw and normalized sequencing datasets including healthy controls and individuals with prostate cancer or type 1 diabetes with or without albuminuria. We focused on kidney-RNAs reviewing literature for DKD-linked miRNAs from kidney tissue, cell culture and uEV/urine experiments. RNAs were analyzed by expression heatmaps, hierarchical clustering and selecting stable mRNAs with normalized counts (>200) and minimal coefficient of variation. Kidney-RNAs were decreased after urine storage at -20 & DEG;C vs. -80 & DEG;C. Isolation workflows captured kidney-RNAs with different efficiencies. Ultracentrifugation captured DKD -linked miRNAs that separated healthy and diabetic macroalbuminuria groups. Eleven mRNAs were stably expressed across the datasets. Hence, pre-analytical choices had variable effects on kidney-RNAs-analyzing kidney-RNAs complemented global correlation, which could fade differences in some relevant RNAs. Replicating prior DKD-marker results and discovery of candidate reference mRNAs encourages further uEV biomarker studies.

Automated summary

Urinary extracellular vesicles (uEV) contain non-invasive RNA biomarkers for genitourinary tract diseases. This study aimed to assess the effects of preanalytical variables on uEV isolates and discover stable reference mRNAs. The researchers analyzed various sequencing datasets and found that urine storage temperature and isolation workflow affected kidney-RNAs, while ultracentrifugation captured DKD-linked miRNAs. They also identified 11 stable mRNAs across the datasets. These findings highlight the importance of considering preanalytical choices in uEV biomarker studies.